Convergent evolution in toxin detection and resistance provides evidence for conserved bacterial–fungal interactions

Significance Bacteria and fungi frequently exist as complex, polymicrobial communities during infection. Reconstructing ecological structure in the laboratory is challenging, and, consequently, the precise molecular mechanisms which underpin microbial interactions remain elusive. Using a preclinical model that mimics the cystic fibrosis lung, we found that the bacterium Pseudomonas aeruginosa detects and defends against a disulfide-containing toxin produced by the fungus Aspergillus fumigatus. In a remarkable example of both convergent evolution of toxin defense and environmental cue sensing across kingdoms, we found that these organisms use the same cue to produce/sense this toxin and the same enzymatic mechanism to protect against toxicity. This finding provides strong evidence for P. aeruginosa exposure to A. fumigatus–produced disulfide-containing toxins in natural environments.

culture supernatants (24 hr) also induce PA4170 expression.Fluorescence was read at 8 hr and normalized to cell density (OD600).The data are representative of three independent experiments performed in triplicate.
Statistical significance was calculated by one-way ANOVA with Dunnett's multiple comparisons test using 'SCFM2' as the control column (**** P < 0.0001).Error bars represent standard deviation from the mean.D. mCherry-tagged PA4170 reporter fluorescence assay examining PA4170 protein expression following growth in spent A. fumigatus culture supernatants grown for 0-24 hr in SCFM.Fluorescence was read at 8 hr and normalized to cell density (OD600).The data are representative of three independent experiments performed in triplicate.Statistical significance was calculated by one-way ANOVA with Dunnett's multiple comparisons test using 'SCFM' as the control column (*** P = 0.0006, **** P < 0.0001).Error bars represent standard deviation from the mean.E. mCherry-tagged PA4170 reporter assay examining PA4170 expression following P. aeruginosa growth in spent A. fumigatus SCFM culture supernatants (24 hr) supplemented with different metals

( 5 µM
FeSO 4 , ZnSO 4 , CuSO 4 , MnSO 4 ) immediately prior to P. aeruginosa addition.Fluorescence was read at 8 hr and normalized to cell density (OD600).The data are representative of three independent experiments performed in triplicate.Statistical significance was calculated by one-way ANOVA with Dunnett's multiple comparisons test using 'SCFM only' as the control column (**** P < 0.0001).Error bars represent standard deviation from the mean.F. Gliotoxin production by A. fumigatus during growth in SCFM2 is repressed by zinc supplementation (10 µM ZnSO 4 ).Gliotoxin was quantified in culture supernatants by LC-MS following 24 hr of A. fumigatus growth.The experiment was performed in triplicate.Statistical significance was determined using a Student's t test (**** P < 0.0001).G. A. fumigatus is zinc limited during growth in SCFM2 and activates expression of the zinc starvation regulated protein Aspf2 and the gliotoxin biosynthetic enzyme GliT.Fluorescence assay examining mCherry-tagged Aspf2 and GliT protein expression during growth in SCFM with or without zinc supplementation (10 µM ZnSO 4 ).Expression was normalized to mCherry expression (+/-10 µM ZnSO 4 ) under the control of the constitutive gpdA promoter.Fluorescence was read at 48 hr.The data are representative of three independent experiments performed in triplicate.Statistical significance was determined using a Student's t test (Aspf2 +/-Zn, GliT +/-Zn) (*** P = 0.0007, **** P < 0.0001).H. mCherry-tagged DnoP reporter fluorescence assay examining DnoP protein expression following P. aeruginosa growth in spent A. fumigatus SCFM culture supernatants (24 h), gliotoxin (30 µM) or SCFM.Fluorescence was read at 8 hr and normalized to cell density (OD600).The data are representative of three independent experiments performed in triplicate.Statistical significance was calculated by one-way ANOVA with Dunnett's multiple comparisons test using 'SCFM' as the control column (**** P < 0.0001).Error bars represent standard deviation from the mean.I. mCherry-tagged PA4170 reporter fluorescence assay examining PA4170 protein expression following growth in spent A. fumigatus SCFM culture supernatants (24 hr) supplemented with 5 µM ZnSO 4 prior to fungal inoculation.Gliotoxin (30 µM) and SCFM wells were also supplemented with 5 µM ZnSO 4 .Fluorescence was read at 8 hr and normalized to cell density (OD600).The data are representative of three independent experiments performed in triplicate.Statistical significance was calculated by one-way ANOVA with Dunnett's multiple comparisons test using 'SCFM' as the control column (**** P < 0.0001).Error bars represent standard deviation from the mean.

Fig. S2 .
Fig. S2. A. Zinc starvation mediated by TPEN (N,N,N′,N′-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine) supplementation activates P. aeruginosa PA4170-mCherry expression in a dose-dependent manner (20-80 µM).Supplementation with 10 µM ZnSO 4 partially reverses PA4170 induction.The data are representative of three independent experiments performed in triplicate.Statistical significance was calculated by one-way ANOVA with Dunnett's multiple comparisons test using 'SCFM' as the control column (**** P < 0.0001).Error bars represent standard deviation from the mean.B. P. aeruginosa wild-type or a ∆PA4170 deletion mutant are not susceptible to bisthiomethylgliotoxin exposure (30 µM).Growth curve carried out in MOPS-succinate.The data are representative of three independent experiments performed in triplicate.C. Deletion of PA4170 has no impact on the ability of P. aeruginosa to grow under TPEN mediated zinc limitation (0-320 µM).Growth curve carried out in SCFM.The data are representative of three independent experiments performed in triplicate.D. Growth of a ∆PA4170 deletion mutant is not impacted upon co-culture with A. fumigatus ∆gliZ when compared to wild type or PA4170 rec (plasmid complemented).Cultures were plated for CFU counts 20 hr after P. aeruginosa addition.The data are representative of three independent experiments performed in quadruplicate.Statistical significance was calculated by one-way ANOVA with Dunnett's multiple comparisons test using 'WT + ∆gliZ' as the control column.Error bars represent standard deviation from the mean.

Fig. S3 .
Fig. S3. A. Growth of a ∆PA4170 deletion mutant is more susceptible to chetomin exposure (30 µM) when compared to the P. aeruginosa wild type.Strains cultured in M9-succinate.The data are representative of two independent experiments performed in triplicate.B. Growth of a ∆PA4170 deletion mutant is more susceptible to chaetocin exposure (30 µM) when compared to the P. aeruginosa wild type.Strains cultured in M9-succinate.The data are representative of two independent experiments performed in triplicate.C. Growth of a ∆PA4170 deletion mutant is more susceptible to holomycin exposure (30 µM) when compared to the P. aeruginosa wild type.Strains cultured in M9-succinate.The data are representative of two independent experiments performed in triplicate.D. Romidepsin exposure (30 µM) has no impact on the growth of P. aeruginosa wild type or ∆PA4170.Strains cultured in M9-succinate.The data are representative of two independent experiments performed in triplicate.E. mCherry-tagged PA4170 reporter assay examining the impact of zinc supplementation (10 µM ZnSO 4 ) on the ability of the ETPs gliotoxin (GT), bisthiomethylgliotoxin (bmGT), chetomin (CHE), chaetocin (CHA), holomycin (HOL) or romidepsin (RHO) to induce PA4170-mCherry expression (all ETPs added at 30 µM).TPEN was added at 50 µM.Fluorescence was read at 8 hr and normalized to cell density (OD600).The data are representative of two independent experiments performed in triplicate.Statistical significance was calculated by one-way ANOVA with Dunnett's multiple comparisons test using 'DMSO' as the control column (**** P < 0.0001).Error bars represent standard deviation from the mean.

Fig. S4 .
Fig. S4.PA4170 orthologs (red boxes) are found in most sequenced P. aeruginosa isolates (279/284) and are interspersed amongst other sequenced Pseudomonas strains (n = 74), including P. fluorescens, P. antarctica, P. tolaasii and P. corrugata.A previously unidentified phylogenetic sub-branch of P. chlororaphis which encodes a PA4170 ortholog is also indicated (orange).Maximum-likelihood tree was assembled with 16s rRNA sequences from representative, sequenced strains across the Pseudomonas genus.

Fig
Fig. S5. A. BLAST search using PA4170 as a query sequence against the Protein Data Bank (PDB) identifies structurally related orthologs of PA4170 in bacteria and fungi-disulfide natural product oxidases (n = 9).Alignments wee visualized with the NCBI Multiple Sequence Alignment Viewer (MSA).The CPYC motif, characteristic of dithiol oxidases is highlighted.B. Sequence identity, coverage, and mismatch of the nine BLAST search PDB matches to PA4170.C. Phylogenetic tree of PA4170 matches as illustrated by BLAST pairwise alignment tree viewer (Fast Minimum Evolution).

Table S1 .
Strains and plasmids used in this study.gliT ORF fused with an ALFA tag linker and C-terminus mCherry.Plasmid contains bleoR (from pAN 8.1); Amp R This study pSKD7 gliT promoter and PA4170 ORF fused with an ALFA tag linker and Cterminus mCherry.Plasmid contains bleoR (from pAN 8.1); Amp R This study pSKD8 aspf2 promoter and aspf2 ORF fused with an ALFA tag linker and Cterminus mCherry.Plasmid contains bleoR (from pAN 8.1); Amp R This study